Alzheimer""s disease affects approximately 20 to 40% of the population over 80 years of age, the fastest growing age group in the United States and other post-industrial countries. Common features in the brain of patients with Alzheimer""s disease include the presence of abundant intraneuronal neurofibrillary tangles (NFTs) and extracellular amyloid rich xcex2-amyloid plaques. NFTs are cytoskeletal pathologies largely composed of aggregates of hyperphosphorylated tau proteins assembled into periodically restricted amyloid fibers called paired helical filaments. The major component of amyloid plaques is a peptide, a small 39-43 aminoacid long xcex2-amyloid peptide that is generated from the cleavage of a larger amyloid precursor protein. However, except for diffuse plaques formed almost exclusively of B-amyloid peptides, amyloid plaques are complex lesions containing numerous associated cellular products. Mutations causing increased production of the 42 amino acid form of this peptide have been genetically linked to autosomal dominant familial forms of Alzheimer""s diseases. Deposits of xcex2-amyloid occur very early in the disease process, long before clinical symptoms develop. Because these mutations appear to be pathogenic and cause Alzheimer""s diseases in transgenic mice, xcex2-amyloids are widely believed to play a causal role in the disease. Whether or not amyloid deposits are causal, they are certainly a key part of the diagnosis. Further, because amyloid plaques occur early in the disease, the ability to image deposits would provide a convenient marker for early diagnosis and prevention of the disease as well as a method for monitoring the effectiveness of therapeutic regimens.
Alzheimer""s disease is currently definitively diagnosed by taking sections from postmortem brain and quantifying the density of neocortical amyloid deposits. Unfortunately, current techniques for detecting amyloid deposits and/or NFTs require postmortem or biopsy analysis. For example, thioflavin fluorescent-labeling of amyloid in brain sections in vitro is currently a widely-used method for evaluation of the brain. Another potential amyloid probe, Chrysamine-G, a congo red derivative, has also been developed. Congo red is a charged molecule and thus lacks sufficient hydrophobicity for diffusion through the blood brain barrier and is therefore not useful as an in vivo label. See Klunk et al, Neurobiology of Aging, 16:541-548 (1995), and PCT Publication No. WO 96/34853. Chrysamine G enters the blood brain barrier better than Congo red, but its ability to label amyloid plaques in Alzheimer""s brain appears weak. See for example, H. Han, C-G Cho and P. T. Lansbury, Jr J. Am. Chem. Soc. 118, 4506 (1996); N. A. Dezutter et al, J. Label. Compd. Radiopharm. 42, 309 (1999). Similarly, earlier attempts to use monoclonal antibodies as probes for in-vivo imaging of xcex2-amyloid were hampered by their limited ability to cross the blood brain barrier. See R. E. Majocha et al, J. Nucl. Med. 33, 2184 (1992). More recently, the use of monobiotinylated conjugates of 1251-Axcex2 1-40 with permeability through the blood brain barrier has also been proposed (See Y. Saito et al., Proc. Natl. Acad. Sci. USA 22, 2288 (1991)), but its ability to label xcex2-amyloid plaques and/or NFTs in vivo has not yet been demonstrated. Quantitation of the deposits in vivo is not yet possible with the currently available probes. Accordingly, a need exists for a convenient marker for early diagnosis of Alzheimer""s disease.
In vivo, non invasive determination of regional cerebral glucose metabolic rates (rCMRG1) with positron emission tomography (PET) has been an important tool in the assessment of brain function in Alzheimer""s disease patients. Numerous studies using 2-[F-18]fluoro-2-deoxy-D-glucose (FDG) have demonstrated a characteristic metabolic pattern of hypometabolism in temporoparietal and frontal association areas. A few of these studies have compared rCMRGI with postmortem regional neuronal pathology. These results and the uncertainties of the Alzheimer""s disease pathogenic cascade highlight the importance of assessing amyloid and neurofibril deposition in vivo, non-invasively in these patients.
The present invention provides methods for labeling structures, including xcex2-amyloid plaques and neurofibrillary tangles, in vivo and in vitro, and comprises contacting a compound of formula (I): 
with mammalian tissue. In formula (I), R1 is selected from the group consisting of xe2x80x94C(O)-alkyl, xe2x80x94C(O)-alkylenyl-R4, xe2x80x94C(O)O-alkyl, xe2x80x94C(O)O-alkylenyl-R4, xe2x80x94Cxe2x95x90C(CN)2-alkyl, xe2x80x94Cxe2x95x90C(CN)2-alkylenyl-R4, 
R4 is a radical selected from the group consisting of alkyl, substituted alkyl, aryl and substituted aryl; R5 is a radical selected from the group consisting of xe2x80x94NH2, xe2x80x94OH, xe2x80x94SH, xe2x80x94NH-alkyl, xe2x80x94NHR4, xe2x80x94NH-alkylenyl-R4, xe2x80x94O-alkyl, xe2x80x94O-alkylenyl-R4, xe2x80x94S-alkyl, and xe2x80x94S-alkylenyl-R4; R6 is a radical selected from the group consisting of xe2x80x94CN, xe2x80x94COOH, xe2x80x94C(O)O-alkyl, xe2x80x94C(O)O-alkylenyl-R4, xe2x80x94C(O)-alkyl, xe2x80x94C(O)-alkylenyl-R4, xe2x80x94C(O)-halogen, xe2x80x94C(O)NH2, xe2x80x94C(O)NH-alkyl, xe2x80x94C(O)NH-alkylenyl-R4; R7 is a radical selected from the group consisting of O, NH, and S; and R8 is N, O or S.
In formula (I), R2 and R3 are each independently selected from the group consisting of alkyl and alkylenyl-R10, wherein R10 is selected from the group consisting of xe2x80x94OH, xe2x80x94OTs, halogen, spiperone, spiperone ketal and spiperone-3-yl. Alternatively, R2 and R3 together form a heterocyclic ring, optionally substituted with at least one radical selected from the group consisting of alkyl, alkoxy, OH, OTs, halogen, alkylenyl-R10, carbonyl, spiperone, spiperone ketal and spiperone-3-yl. In the compounds of formula (I), one or more of the hydrogen, halogen or carbon atoms can, optionally, be replaced with a radiolabel.
For in vitro detection of xcex2-amyloid plaques and neurofibrillary tangles in brain tissue, the plaques are labeled, and the brain tissue is then observed with a fluorescence microscope. For in vivo detection, the xcex2-amyloid plaques and neurofibrillary tangles in brain tissue are labeled, preferably by injection of a solution containing a radiolabeled compound of formula (I). The locations of the labeled xcex2-amyloid plaques and neurofibrillary tangles are then observed by any method capable of detecting and depicting the distribution of the radiolabeled compound within the body.
According to the methods of the invention, amyloid deposits in cryostat and paraffin sections of Alzheimer-diseased (AD) brain tissue are labeled with a level of sensitivity similar to thioflavin S. Use of the present invention, however, has several advantages over using thioflavin S. Namely, no pretreatments are required. Moreover, unlike with thioflavin S, the methods work with minimal washing and without formalin or paraformaldehyde fixation or differentiation of tissue. Additionally, stock solution can be kept in the freezer for six months and still produce acceptable results at 1/100 to 1/1,000 dilutions, eliminating the need to make the stock up fresh, as is required for thioflavin S labeling.
Systemically injected compositions according to the invention readily penetrate the blood brain barrier and label amyloid deposits and neurofibrillary tangles demonstrating the ability of the present compositions to act as an in vivo imaging probe. The methods of the invention achieve in vivo labeling and detection of xcex2-amyloid plaques and neurofibrillary tangles in the brain of a living patient. The methods of the invention not only permit detection of Alzheimer""s disease, but also provide a way for physicians to monitor the progress of patients undergoing treatment for the disease. Thus, physicians can better determine whether a particular treatment method is successful and worthwhile.
In still another embodiment, the invention is directed to a composition comprising a compound of formula (I): 
R1 is selected from the group consisting of xe2x80x94C(O)-alkyl, xe2x80x94C(O)-alkylenyl-R4, xe2x80x94C(O)O-alkyl, xe2x80x94C(O)O-alkylenyl-R4, xe2x80x94Cxe2x95x90C(CN)2-alkyl, xe2x80x94Cxe2x95x90C(CN)2-alkylenyl-R4, 
R4 is a radical selected from the group consisting of alkyl, substituted alkyl, aryl and substituted aryl; R5 is a radical selected from the group consisting of xe2x80x94NH2, xe2x80x94OH, xe2x80x94SH, xe2x80x94NH-alkyl, xe2x80x94NHR4, xe2x80x94NH-alkylenyl-R4, xe2x80x94O-alkyl, xe2x80x94O-alkylenyl-R4, xe2x80x94S-alkyl, and xe2x80x94S-alkylenyl-R4; R6 is a radical selected from the group consisting of xe2x80x94CN, xe2x80x94COOH, xe2x80x94C(O)O-alkyl, xe2x80x94C(O)O-alkylenyl-R4, xe2x80x94C(O)-alkyl, xe2x80x94C(O)-alkylenyl-R4, xe2x80x94C(O)-halogen, xe2x80x94C(O)NH2, xe2x80x94C(O)NH-alkyl, xe2x80x94C(O)NH-alkylenyl-R4; R7 is a radical selected from the group consisting of O, NH, and S; R8 is N, O or S; R2 is selected from the group consisting of alkyl and alkylenyl-R5 and R3 is alkylenyl-R5, and R5 is selected from the group consisting of xe2x80x94OH, xe2x80x94OTs, halogen, spiperone, spiperone ketal, and spiperone-3-yl, or R2 and R3 together form a heterocyclic ring, optionally substituted with at least one radical selected from the group consisting of alkyl, alkoxy, OH, OTs, halogen, alkylenyl-R5, carbonyl, spiperone, spiperone ketal and spiperone-3-yl. One or more of the hydrogen, halogen or carbon atoms can optionally be replaced with a radiolabel.
The invention is more preferably related to a composition comprising a compound of formula (II): 
R2 is selected from the group consisting of alkyl and alkylenyl-R10 and R3 is alkylenyl-R10, wherein R10 is selected from the group consisting of xe2x80x94OH, xe2x80x94OTs, halogen, spiperone, spiperone ketal and spiperone-3-yl, or R2 and R3 together form a heterocyclic ring, optionally substituted with at least one radical selected from the group consisting of alkyl, alkoxy, OH, OTs, halogen, alkylenyl-R10, carbonyl, spiperone, spiperone ketal and spiperone-3-yl, and R9 is an alkyl, aryl or substituted aryl group, and to pharmaceutically acceptable salts and solvates thereof. One or more of the hydrogen, halogen or carbon atoms can optionally be replaced with a radiolabel.